Journal: eLife

Article Title: Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

doi: 10.7554/eLife.65620

Figure Lengend Snippet: HEK293T cells expressing the components of the bioluminescence resonance energy transfer (BRET) biosensor for free Gβγ (top) or Gαi-GTP (bottom), the membrane-anchored FRB construct, and the indicated FKBP-fused G-protein regulators GIV GBA, AGS1*, Ric-8A*, or R12 GL were stimulated with rapamycin (0.5 μM) at the indicated time during kinetic BRET measurements. Stimulation of ectopically expressed M4 muscarinic receptor (M4R) with carbachol (100 μM) was done as a reference condition, and rapamycin stimulation of cells not expressing FKBP-fused constructs was done as a negative control. Bar graphs on the right summarize the BRET changes 90 s after addition of rapamycin or carbachol. Mean ± SD, n = 3–4. In the kinetic traces, the SD is displayed as bars of lighter color tone than data points and only in the positive direction for clarity. Figure 2—source data 1. Numerical data used for the upper panel (free Gβγ biosensor). Figure 2—source data 2. Numerical data used for the lower panel (Gαi-GTP biosensor).

Article Snippet: The plasmids encoding FKBP-fused constructs were generated by replacing the pseudojanin sequence between the NruI/BamHI sites of pmRFP-FKBP-pseudojanin (Addgene, #37999) by different sequences: for pmRFP-FKBP-GIV GBA it was human GIV amino acids 1660–1705 ( ); for pmRFP-FKBP-AGS1* it was full length rat AGS1 (aka DEXRAS) bearing a C278S mutation to disrupt its CAAX box that allows membrane targeting; for pmRFP-FKBP-Ric-8A* it was rat Ric-8A amino acids 12–492 ( ; ); for pmRFP-FKBP-R12 GL it was mouse RGS12 amino acids 1185–1221 ( ); and for pmRFP-FKBP-GIV-CT it was human GIV amino acids 1660–1870 ( ).

Techniques: Expressing, Bioluminescence Resonance Energy Transfer, Construct, Negative Control

Journal: eLife

Article Title: Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

doi: 10.7554/eLife.65620

Figure Lengend Snippet:

Article Snippet: The plasmids encoding FKBP-fused constructs were generated by replacing the pseudojanin sequence between the NruI/BamHI sites of pmRFP-FKBP-pseudojanin (Addgene, #37999) by different sequences: for pmRFP-FKBP-GIV GBA it was human GIV amino acids 1660–1705 ( ); for pmRFP-FKBP-AGS1* it was full length rat AGS1 (aka DEXRAS) bearing a C278S mutation to disrupt its CAAX box that allows membrane targeting; for pmRFP-FKBP-Ric-8A* it was rat Ric-8A amino acids 12–492 ( ; ); for pmRFP-FKBP-R12 GL it was mouse RGS12 amino acids 1185–1221 ( ); and for pmRFP-FKBP-GIV-CT it was human GIV amino acids 1660–1870 ( ).

Techniques: Western Blot, Recombinant, Plasmid Preparation, Mutagenesis, Expressing, Variant Assay, Luciferase

Journal: Journal of Bone and Mineral Research

Article Title: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk

doi: 10.1002/jbmr.120

Figure Lengend Snippet: GR-specific gene silencing impaires osteo/adipogenesis capacity of hBMSCs. ( A ) Internalization of the glucocorticoid receptor in response to dexamethasone (Dex). hBMSCs were incubated in the presence of 1 µM Dex for 24 hours. (GR green , nuclei blue ). ( B ) Western blot analysis of GR expression after specific knock-down in hBMSCs transduced with pLV-GRi. Control: pLVTHM empty vector (pLV-emp). Sam68 was used as a loading control. Effects of GR silencing on hBMSC adipogenesis ( C ) and osteogenesis ( D ). Analysis of GFP expression by flow cytometry. hBMSCs transduced with pLVTHM were harvested, and GFP expression was analyzed by flow cytometry ( white ). Negative control: noninfected hBMSCs ( gray ). hBMSCs transduced with either the empty or GRi-containing vector were induced to differentiate in the presence of adipogenic medium (AM) or osteogenic medium (OM). ( C ) Percentage of mature adipocytes stained with Nile red was estimated by flow cytometry. Percentage of GFP + and GFP – mature adipocytes after the induction period are represented by bars. Results show the mean ± SD of triplicate samples. Significance of the difference between GFP + and GFP – in pLV-GRi cells was determined using Student's t test; ** p < .01. ( D ) Osteogenic induction of transduced hBMSCs. Cells were stained with alizarin red S. Control: absence of OM. Alizarin red calcium quantification is shown in Supplemental Fig. 2 A .

Article Snippet: The H1-shRNA expression cassette then was excised and cloned into the lentiviral pLVTHM plasmid using EcoRI-ClaI sites (Addgene Plasmid 12247, http://www.addgene.org ). c-Jun cDNA was purchased from imaGenes (Clone IRATp970B0488D6, http://www.imagenes-bio.de ), related to the NCBI c-Jun sequence NM_000228. c-Jun cDNA was excised and cloned into the pSP72 vector (Promega Plasmid P2191, http://www.promega.com ) using EcoRI and BamH1.

Techniques: Incubation, Western Blot, Expressing, Knockdown, Transduction, Control, Plasmid Preparation, Flow Cytometry, Negative Control, Staining

Journal: Journal of Bone and Mineral Research

Article Title: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk

doi: 10.1002/jbmr.120

Figure Lengend Snippet: c-Jun expression level is involved in hBMSC proliferation and influences hBMSC differentiation capacity. ( A ) hBMSCs were transduced at different MOIs with the bicistronic pWPI-c-Jun cDNA or pLV-c-Juni RNAi expression vector, and GFP expression was analyzed by flow cytometry. Western blot analysis of c-Jun expression after specific knockdown or overexpression. Transduced cells (100% infected) were analyzed for protein expression by Western blot using a specific c-Jun antibody. Control: hBMSCs transduced with the empty vector (pLV-emp). Sam68 was used as a loading control. Protein quantification is shown in Supplemental Fig. 7. ( B ) c-Jun expression regulates hBMSC proliferation. hBMSCs showing approximately 50% infection efficiency (with either pWPI-c-Jun or pLV-c-Juni vector) were plated at 2000 cells/cm 2 , analyzed for GFP expression weekly, and replated at the same cell concentration until the end of the experiment. Control: cells transduced with the specific empty vector (pWPI or pLVTHM empty vector). Results show the mean ± SD of triplicate samples. pWPI-c-Jun and pLV-c-Juni were significantly different from the empty vector at all indicated time points. ** p < .01, * p < .05. ( C ) c-Jun overexpression inhibits adipogenic capacity of hBMSCs. Percentage of mature adipocytes from GFP – and GFP + cells (cells transduced with the empty and c-Jun-cDNA expressing vectors, respectively) after the induction period are represented. c-Jun overexpression stimulates the osteogenic capacity of hBMSCs. Formation of mineralized extracellular matrix in hBMSCs transduced with either the empty vector (pWPI-empty) or the vector expressing c-Jun cDNA (pWPI-c-Jun), visualized by alizarin red S staining, is shown ( lower panel ). Significance of the difference between GFP + and GFP – in pWPI-c-Jun cells was determined using Student's t test; * p < .05. ( D ) c-Jun knockdown rescues adipogenesis from the inhibition caused by PDGF. hBMSCs transduced with either the empty (pLV-emp) or c-Juni-expressing vector (pLV-c-Juni) were induced to differentiate to adipogenic lineages in the presence or absence of PDGF (25 ng/mL). c-Jun knockdown inhibits hBMSC osteogenic differentiation. hBMSCs transduced with the pLV-c-Juni vector reduced the quantity of mineralized extracellular matrix extensively compared with control cells (pLV-empty). Results show the mean ± SD of triplicate samples. Significance of the difference between pLV-empty GFP + cells in the presence/absence of PDGF (* p < .05) and in the presence or absence of c-Jun (** p < .01) was determined using Student's t test.

Article Snippet: The H1-shRNA expression cassette then was excised and cloned into the lentiviral pLVTHM plasmid using EcoRI-ClaI sites (Addgene Plasmid 12247, http://www.addgene.org ). c-Jun cDNA was purchased from imaGenes (Clone IRATp970B0488D6, http://www.imagenes-bio.de ), related to the NCBI c-Jun sequence NM_000228. c-Jun cDNA was excised and cloned into the pSP72 vector (Promega Plasmid P2191, http://www.promega.com ) using EcoRI and BamH1.

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Western Blot, Knockdown, Over Expression, Infection, Control, Transduction, Concentration Assay, Staining, Inhibition

Journal: Journal of Bone and Mineral Research

Article Title: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk

doi: 10.1002/jbmr.120

Figure Lengend Snippet: c-Jun expression level is involved in hBMSC proliferation and influences hBMSC differentiation capacity. ( A ) hBMSCs were transduced at different MOIs with the bicistronic pWPI-c-Jun cDNA or pLV-c-Juni RNAi expression vector, and GFP expression was analyzed by flow cytometry. Western blot analysis of c-Jun expression after specific knockdown or overexpression. Transduced cells (100% infected) were analyzed for protein expression by Western blot using a specific c-Jun antibody. Control: hBMSCs transduced with the empty vector (pLV-emp). Sam68 was used as a loading control. Protein quantification is shown in Supplemental Fig. 7. ( B ) c-Jun expression regulates hBMSC proliferation. hBMSCs showing approximately 50% infection efficiency (with either pWPI-c-Jun or pLV-c-Juni vector) were plated at 2000 cells/cm 2 , analyzed for GFP expression weekly, and replated at the same cell concentration until the end of the experiment. Control: cells transduced with the specific empty vector (pWPI or pLVTHM empty vector). Results show the mean ± SD of triplicate samples. pWPI-c-Jun and pLV-c-Juni were significantly different from the empty vector at all indicated time points. ** p < .01, * p < .05. ( C ) c-Jun overexpression inhibits adipogenic capacity of hBMSCs. Percentage of mature adipocytes from GFP – and GFP + cells (cells transduced with the empty and c-Jun-cDNA expressing vectors, respectively) after the induction period are represented. c-Jun overexpression stimulates the osteogenic capacity of hBMSCs. Formation of mineralized extracellular matrix in hBMSCs transduced with either the empty vector (pWPI-empty) or the vector expressing c-Jun cDNA (pWPI-c-Jun), visualized by alizarin red S staining, is shown ( lower panel ). Significance of the difference between GFP + and GFP – in pWPI-c-Jun cells was determined using Student's t test; * p < .05. ( D ) c-Jun knockdown rescues adipogenesis from the inhibition caused by PDGF. hBMSCs transduced with either the empty (pLV-emp) or c-Juni-expressing vector (pLV-c-Juni) were induced to differentiate to adipogenic lineages in the presence or absence of PDGF (25 ng/mL). c-Jun knockdown inhibits hBMSC osteogenic differentiation. hBMSCs transduced with the pLV-c-Juni vector reduced the quantity of mineralized extracellular matrix extensively compared with control cells (pLV-empty). Results show the mean ± SD of triplicate samples. Significance of the difference between pLV-empty GFP + cells in the presence/absence of PDGF (* p < .05) and in the presence or absence of c-Jun (** p < .01) was determined using Student's t test.

Article Snippet: EcoRV and PvuII were used to get c-Jun cDNA from pSP72, and it was cloned in the PmeI cassette of pWPI plasmid (Addgene Plasmid 12254). c-Jun cDNA as well as GR and c-Jun shRNA sequences were checked after cloning in pWPI and pLVTHM vectors to confirm 100% correspondence with the original sequences.

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Western Blot, Knockdown, Over Expression, Infection, Control, Transduction, Concentration Assay, Staining, Inhibition